Abstract


BACKGROUND: Cord blood stem cells are one of ideal target cells for gene therapy, but low gene transferring rate is the main difficulty at recent. Janus kinase tyrosine 2 (JAK2) plays an important role in self-renewing of cord blood stem/progenitor cell12s. Therefore, cord blood CD34+ cell line modified by target-amplified JAK2 genes has been developed yet by using gene regulating expression technique in order to overcome low transferring rate of cord blood genes.
OBJECTIVE: To investigate the feasibility and reliability of a long-term amplified regulation for cord blood stem/progenitor cells mediated by transgene JAK2.
SETTING: Department of Hematology, Beijing Hospital, Ministry of Health.
MATERIALS: The experiment was carried out in the Laboratory of Hematological Department, Beijing Hospital, Ministry of Health from June 2003 to April 2006. Cord blood was derived from umbilical cord which was immediately cut from healthy, full-term and natural-parturition infants and was provided by Department of Obstetrics & Gynecology, Beijing Hospital. The experiment was approved by the local ethical committee, and informed consent was obtained from expectant mothers and their relatives for the use of cord blood cells. MiniMACS magnetic separation apparatus and immunomagnetic beads adsorbing CD34 single antibody were provided by Miltenyi Biotec Company, Germany; flow cytometer by FACScalibur, USA; recombinant human stem cell factor (rhSCF), Flt3 ligand (FL), human interleukin-6 (hIL-6), granulocyte macrophage colony stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF) and thrombopoeitin (TPO) by PeproTec Company; nude mice of the SPF level by Animal Center of Beijing Medical University.
METHODS: Retroviral vector MGI-F2JAK2, which was composed of functional catalytic domain of JAK2 genes and two site proteins (2xF36v, F2) combined with synthetic drug (AP20187) of target gene of small molecules, was constructed. AP20187 might specially combine with F36v to cause dimerization of JAK2 so as to activate signal conduction in cells. In addition, the vector included green fluorescence protein reporter gene, which was regarded as a label to detect proliferation. MiniMACS magnetic separation apparatus was used to purify and separate cord blood CD34+ cells. While, retrovirus supernatant including JAK2 was used to transfer cord blood CD34+ cells. After transduction, CD34+ cells were cultured with stem cell factor (SCF), Flt3 ligand, TPO and IL-6 and divided into control group (not adding AP20187) and experimental group (AP20187).
MAIN OUTCOME MEASURES: ① Flow cytometer was used to detect percentage of green fluorescence protein reporter gene in the CD34+ cells and to determine gene transferring rate. ② Colony culture results of cord blood stem/progenitor cells after amplification. ③ Nude mice were given subcutaneous injection of ten-week cultured cord blood CD34+ cells at costa and neoplasia was observed after 30 days.
RESULTS: ① Plentiful amplification of CD34+ cells was observed in both experimental group and control group. With the culture time passing by, positive rate of gel-filtered platelet of amplified CD34+ cells in the experimental group was gradually increased based on the basic level and more than 95% in the 11th week; however, positive rate of green fluorescence protein reporter gene in the control group was gradually decreased below the basic level and disappeared finally. ② Transgenic CD34+ cells in the experimental group still could generate brust forming unit-erythroid (BFU-E), colony-forming units granulocute/monocyte (CFU-GM) and multipotential hematopoietic progenitors (CFU-Mix); especially, CFU-GM was the main cell in hemopoietic progenitor cell (HPC). ③ Nude mice did not have neoplasia.
CONCLUSION: Human cord blood CD34+ cells of transferring JAK2 genes may cooperate with other cytokines to amplify cord blood stem/progenitor cells in vitro for long. Therefore, this is potentially valuable for stem cells to treat some hereditary hematologic disease.

INTRODUCTION

Cord blood stem cells are rich in resources and difficult to be polluted by virus or tumor, which are considered as low immunogenicity and few xenogenous rejections. Therefore, it becomes a new material for the transplantation of stem cells except bone marrow and peripheral blood. Cord blood stem cells have the abilities of strong self-renewing, multipotential differentiation and reconstructed haemopoiesis. So they are one of ideal target cells for gene therapy. But low transferring rate of genes and unstable expression and regulation are the main difficulties for gene therapy at present[1]. This study was designed to establish gene expression-regulation technique and construct a clone of retroviral vector containing JAK2. The vector was transferred into cord blood stem cells so as to induce dimerization of JAK2 via synthetic drug (AP20187) of target gene of small molecule, start signal conduction and observe whether a long-term amplification and regulation of transgenic cord blood stem cells could be realized.

MATERIALS AND METHODS

Materials
The experiment was carried out in the Laboratory (ministerial level) of Hematological Department, Beijing Hospital of Ministry of Public Health from June 2003 to April 2006. Cord blood was derived from umbilical cord which was immediately cut from healthy, full-term and natural-parturition infants and was provided by Department of Obstetrics & Gynecology, Beijing Hospital. The experiment was approved by the local Ethical Committee, and informed consent was obtained


from expectant mothers and their relatives for the use of cord blood cells. MiniMACS magnetic separation apparatus and immunomagnetic beads adsorbing CD34 single antibody were provided by Miltenyi Biotec Company, Germany; flow cytometer by FACScalibur, USA; recombinant human stem cell factor (rhSCF), Flt3 ligand (FL), human interleukin-6 (hIL-6), granulocyte macrophage colony stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF) and thrombopoeitin (TPO) by PeproTec Company; single cloning antibody PE-CD34 and FITC-CD38 by BD PharMingen; nude mice of the SPF level by Animal Center of Beijing Medical University.

Methods
Cell source and separation and purification of cord blood CD34+ cells
Cord blood was anti-coagulated with heparin, diluted with PBS, settled with bagolax, gradiently centrifuged with Ficoll (relative density: 1.077) to separate mononuclear cell (MNC). After washing, antibody and magnetic beads antibody (anti-CD34 antibody: QBEND/10; magnetic beads: caprine-anti-mouse IgG1 immune magnetic beads; dilution ratio: 1:5) were labeled. Negative CD34 cells are isolated from CD cells by using MiniMACS magnetic separation apparatus. In addition, detached dowel was moved out of the magnetic field, and then pressed and eluted to collect CD34+ cells. Flow cytometer was used to measure the purity. The labeled antibody was PE-combined anti-CD34 antibody.

Construction of JAK2 retroviral vector and establishment of packaging cell line
Retroviral vector was constructed as the reference[2]. The vector was a MSCV-based retroviral vector and was composed of functional catalytic domain of JAK2 genes and two site proteins (2xF36v, F2) combined with synthetic drug (AP20187) of target gene of small molecules; meanwhile, the vector contained green fluorescence protein reporter gene, which was regarded as a label to detect proliferation. F36v was derived from endogenous FKBP protein whose phenylalanine at the 36th site was replaced by valine in order to prevent amplification of non-transgene induced by non-specific combination between AP20187 and endogenous FKBP protein. The carrier was proved with restriction enzyme and sequence and named as MGI-F2JAK2 (Figure 1).

The establishment of GPE+86 cell strain was detailed in reference[2]; while, transferring in the virus supernatant was colleted to transfer PG13-packed cell line, and the detailed operations were recorded in the reference[3]. PG13 cell strain clone which had the strongest toxicity was used for the transduction cord blood CD34 cells through transfection of HT1080 cells and GFP expression.

Gene transfer and in vitro amplification of JAK2 transgenic cord blood CD34+ cells
Cord blood CD34+ cells with the purity of 91%-95% was incubated with IMDM culture media containing 20% Bit9500 (Stem Cell Technologies), 100 g/L low density lipoprotein (LDL, Sigma Company), 10 μg/L hIL-6, 50 μg/L hSCF, 20 μg/L FL and 50 μg/L TPO and stimulated for 48 hours, and then with IMDM culture medium containing 10% FBS and the same concentration of above mentioned cytokines, supplemented with JAK2-expressing virus supernatant of retroviral vector PG13 cell strain. The cells were cultured in 6-well culture plate with CH296 Retronectin (Takara Biomedicals Company) for 72 hours. Virus supernatant was changed every 24 hours for three times in total. Cord blood CD34+ cells after JAK2 gene transfer were centrifuged and thoroughly washed twice so as to clear cytokines and serum. And then, cells were re-suspended in 10% FCS culture system and divided into experimental group (AP20187+SCF+FL+TPO+IL-6, ASFTI group) and control group (SCF+FL+TPO+IL-6, SFTI group; not adding AP20187). The final concentration of AP20187 was 100 nmol (TPO 1.0×104 U/L, hIL-6 10 μg/L, SCF 50 μg/L, FL 50 μg/L). Cells were cultured in incubator with 5% CO2 saturation humidity at 37 ℃ for a long period. Fresh culture media and cytokines were changed every week and GFP expression was measured at the same time. Before gene transfection, the beginning amount of CD34+ cells ranged from 3.5×104 to 5.6×105.

Cell phenotype by using flow cytometer
After gene transfer of CD34+ cells, flow cytometer (FACScalibur) was used to measure percentage of green fluorescence protein (GFP) in CD34+ cells so as to determine gene transferring rate, and GFP expressing rate at day 0 after gene transfer was regarded as base line. GFP expression of CD34+ cells was measured regularly in every week. Monoclonal antibody-labeled PE-CD34/FITC-CD38 double labeling technique was used to label cord blood cells in the experimental and control groups; meanwhile, negative IgG homotype control was designed, diluted with antibody (1:5), incubated at 4℃ for 30 minutes and washed with PBS twice. PBS/BSA/Azide was cooled and re-suspended on flow cytometer to regularly analyze phenotype changes of cellular immunity by using CELLQestv3.1 software.

Demi-solid colony culture of stem/progenitor cells after cord blood culture
Cord blood CD34+ cells at 10 weeks after culture were washed to clear cytokines and AP20187, maintained in IMDM culture medium containing 1.2% bagolax, 30% PBS, 1% BSA and 5×10-4 mol/L 2-mercaptoethanol with 2 mL for each, and put in three 30-mm culture mediums with 0.5 mL for each. The concentration of cell was 2.5×106 /L. 20 μg/L hIL-3, 50 μg/L SCF and 5×103 U/L Epo were added in the above-mentioned culture medium to measure burst-forming units erythroid (BFU-E), and the colony was counted on the 14th day; 40 μg/L GM-CSF, 20 μg/L hIL-3 and 50 μg/L SCF were added in the culture medium to measure colony-forming units granulocute/monocyte (CFU-GM), and the colony was counted on the 8th day; 50 μg/L SCF, 3 mg/L Epo, 40 μg/L GM-CSF and 20 μg/L IL-3 were added in the culture medium to detect multipotential hematopoietic progenitors (CFU-Mix), and the colony was counted on the 14th day. All colonies were cultured in incubator with 0.05 volume fraction of CO2 and saturation humidity at 37 ℃ and observed under inverted microscope.

Chromatosome karyotype analysis
At 10 weeks of culture, 1×105 cord blood CD34+ stem/progenitor cells was incubated in demecolcine (Sigma) with the final concentration of 2.5 mg/L for 3 hours, dealt with KCI at 37 ℃ for 30 minutes, centrifuged to clear supernatant, added with methyl alcohol, fixed with acetic acid glacial for 30 minutes, re-fixed, made in suspension dribbling sections, dried, stained with 10% Giemsa and observed under microscope.

Induced neoplasia test in nude mice
Female BALB/c nude mice of 4-6 weeks old and weighing 20 g provided by the Animal Center of Peking Medical University were fed in laminar flow of SPF level. 5×106/ 0.2 mL cord blood CD34+ cells at 10 weeks after culture were subcutaneously injected into flank of mice, and neoplasia was observed 30 days later.

Statistical analysis
Experimental data were expressed as Mean±SD and analyzed with t test. SPSS 11.0 software was used by the first and second authors in this study.

RESULTS

In vitro amplification of transgene cord blood CD34+ cells and dynamic changes of GFP
Retroviral vector MGI-F2JAK2 constructed in this study might code to generate GFP. When the vector was moved in CD34+ cells, flow cytometer was used to measure GFP expression so as to determine gene transferring rate, which was (49.3±6.2)% (n=4). Growth of transgene cord blood stem/progenitor cells and response to AP20187 synthesized by small molecule target genes were dynamically observed through measuring positive percentage of GFP in CD34+ cells. GFP expression in cord blood CD34+ cells was detected every week and drawn a reactive curve (Figure 2). With the culture time passing by, positive percentage of GFP in CD34+ cells of the experimental group was gradually increased and reached peak at the 11th week. Almost GFP showed 100% positive expression. This suggested that cord blood CD34+ cells of transgene JAK2 were amplified and gained advantage, and the growth was superior to that of non-transgene JAK2. Therefore, non-transgene cells were inhibited and disappeared gradually. The culture in the experimental group might last for more than 12 weeks. Positive GFP cells reduced gradually in the control group and disappeared at the 8th week. This suggested that JAK2 transgene cells were not positively amplified if AP20187 was not used, and over-proliferation of non-transgene cells could inhibit transgene cells. There was significant difference in positive rate of GFP between experimental group and control group (P < 0.01).

Labeling changes of cell subgroup surface after amplification of transgene CD34+ cells
Flow cytometer could dynamically detect changes of important subgroups of CD34+ CD38+ and CD34+ CD38- after amplification culture in cord blood CD34+ cells. In the experimental group, subgroups of CD34+ CD38+ and CD34+ CD38- were amplified (228.26±32.31) and (321.48±40.52) times, respectively at the 6th week. In the control group, subgroups of CD34+ CD38+ and CD34+ CD38- were amplified (146.26±24.28) and (202.62±26.73) times, respectively. There was significant difference between them (P < 0.05, P < 0.01, Figure 3).

Colony culture of transgene cord blood CD34+ cells
Amplified CD34+ cells in the experimental group could still generate BFU-E, CFU-Mix and especially CFU-GM. The colony amount was decreased as compared with that at the 5th week of culture. This suggested that colony formation was gradually slowed with the amplification time passing by (Table 1).

Evaluation of cellular reliability after amplification
At 10 weeks after culture of amplified CD34+ cells in the experimental group, 10 chromatosome karyotypes in the splitting metaphase were randomly analyzed, and there was no abnormality. Meanwhile, when amplified CD34+ cells were inoculated in subcutaneous tissue of BALB/c nude mice which were cultured in sterility condition and SPF level for 30 days, tumor was not observed.

DISCUSSION

Low gene transferring rate of hemopoietic stem cells is an important difficulty for gene therapy of stem cells. If gene transferring rate reaches the standard of gene therapy, positive screening may be used in transgenic stem cells. For example, drug-resistant gene may put in stem cells to generate an ability against toxicity of chemotherapeutic drugs and get rid of those cells without transfection drug resistance so as to screen transgenic stem cells for gene therapy[1,3-4]. However, this method has a lot of difficulties and limitations. For instance, chemotherapeutic drugs may cause severe toxicity to bodies; transgenic stem cells are hard to be directly regulated and easy to be exhausted in an early phase. Recently, in the new method, target gene connects to one or two reversible dimerization binding site protein and is activated through small molecule dimerization compound (AP20187) so as to regulate growth gene transferring cells but not affect non-transfection gene cells. Therefore, target genes are considered as precedent growth based on synthetic drug of small molecule target genes, and cell amplification is restricted to transgenic stem cells[5-7]. This important achievement accords to cell && molecular biological principle and protein engineering[8-9], and even deep investigation of pathway of cell signal conduction in recent years.
Based on cell & molecular biology, dimerization or oligomerization of protein in cell is the core of signal conduction pathway, which can control proliferation, differentiation and apoptosis[7]. In recent years, protein tyrosine kinase (PTK) JAK2 plays an important role in self-renewing and duplication of hemopoietic stem cells based on deep researches on structure and function of PTK and conduction pathway of Janse kinase-signal transducer activator of transcriptions (JAKs-STATs)[10]. Signals of most cytokines are conducted via JAK2. Dimerization of JAK2 may cause activation of downstream signal STATs so as to start proliferation and duplication of cells. We construct retroviral vector (MGI-F2JAK2) containing PTK JAK2[2], and this vector has two dimerization binding sites (F36v) which connect to JAK2. Small molecule dimerization compound (AP20187) has high affinity to binding sites and can cause dimerization of JAK2 to activate signal conduction pathway through binding with F36v. AP20187 is considered as transmembrane permeability and asepsis and has safely used in researches in vivo[5,11]. GFP in this vector is modified with basic radicals and reformed in humanization; meanwhile, it is considered as no species-specificity and widely used as detecting marker for gene transfer[12]. Flow cytometer is used to directly measure transfection efficiency and growth of cells.
Cord blood CD34+ cells are regarded as target cells. JAK2 retroviral vector is transferred into purified cord blood CD34+ cells and induced dimerization through small molecule synthetic drug AP20187; meanwhile, both of them act SCF, FL, TPO and IL-6 in an early phase to realize larger amplification in vitro of cord blood stem/progenitor cells for a long period. With the culture time passing by, positive percentage of GFP in cord blood CD34+ cells is affected by AP20187 obviously. In the control group (cytokines alone), GFP percentage is gradually decreased. At the fourth week, GFP percentage of positive CD34 cells decreases from (49.3±6.2)% to 5%-20% and completely disappears at the 10th week. Oppositely, in the experimental group (with AP20187), positive percentage of GFP in the cord blood CD34+ cells gradually increases from the 1st week, reaches the peak at the 11th week, and is closed to 100% positive expression. This suggests that transgenic cells gain precedent growth through activating JAK2 signals by AP20187. In the aspect of immunophenotype, CD34+ cell subgroup in both experimental group and control group has amplified obviously; however, amplified times of CD34+CD38- and CD34+CD38- cells in the experimental group are significantly different from those in the control group. Researches demonstrate that CD34+CD38- cell subgroup is stem cells in an earlier phase and has a strong proliferation in vivo[13]. Amplification of this cell group is value for application of stem cell transplantation. Otherwise, at 12 weeks after amplification, transgenic cord blood CD34+ cells still have the ability to form progenitor cell colony, and this suggests that amplified cells are still considered as self-renewal and multiple potentialities.
In order to increase gene transferring rate of purified CD34+ cells, we use Bit9500 and CH296 Retronectin in gene conduction culture system. Stem cell cytokines including SCF, FL and TPO may act on stem cells in an early phase[10,14-16]. The combination can synergistically stimulate proliferation of stem cells in an early phase and increase gene transferring rate of viral vector. Security is constantly first question and prerequisite for gene therapy. Phenylalanine at the 36th site of FKBP is replaced by valine in this study in order to avoid the combination between AP20187 and endogenous FKBP. Therefore, the amplified cells are specific and restricted to transgenes but not affect functions of other cells. We detect chromosome of cord blood CD34+ cells which are amplified for 10 weeks, and the results indicate that there is no abnormality of karyotype. In addition, amplified cord blood stem cells do not cause oncogenicity in nude mice. Security and effectiveness of viral vector have been deep researched by a lot of scholars[17].
Results in this study demonstrated that transgenic cord blood stem cells can regulate amplification in vitro and obtain effectively stable transferring rate. This achievement is potentially value for gene therapy. We firmly believe that improvement of gene transfection rate, amplification and regulation of transgenic cells and stable expression of target gene after transfection may make sure gene therapy of cord blood stem cells effectively and widely use in clinic. Based on above-mentioned achievements, gene therapy plays a perspective role in severe immune defect, genetic disease, malignant tumor, protection of hematopoietic stem cells and AIDS.

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靶基因调控的脐血干/祖细胞体外长期扩增与调控**★

赵声明1，彭明婷2，顾惜春1，常乃柏1
1卫生部北京医院血液科，北京市 100730；2卫生部临床检验中心血液室，北京市 100730
赵声明★，男，1965年生，山东省诸城市人，汉族，1988年山东大学医学院毕业, 硕士，副主任医师，主要从事干细胞生物学、细胞信号转导及基因治疗研究。北京市自然科学基金资助项目(7042055)*；首都医学发展基金重点资助项目(2003-2017)*

摘要

背景：脐血干细胞是基因治疗最理想的靶细胞之一，但基因转移率低下是目前面临的主要障碍。酪氨酸激酶JAK2在造血干/祖细胞自我更新中扮演着重要的作用，为了克服脐血基因转移率低下的障碍，根据基因调控表达技术原理，是否可开发一个可以靶向扩增JAK2基因修饰的脐血CD34+细胞体系。
目的：探讨转基因JAK2介导的脐血干祖细胞长期扩增调控的可行性和安全性。
单位：卫生部北京医院血液科。
材料：实验于2003-06/2006-04在卫生部北京医院血液科实验室完成。脐血取自健康、足月、自然分娩后立即断脐的脐血。脐血由北京医院妇产科提供，产妇及家属均知情同意，实验经医学伦理委员会批准。MiniMACS磁性分离仪、免疫磁珠吸附CD34单抗购自德国Miltenyi Biotec公司， 流式细胞仪购自美国FACScalibur，人重组干细胞因子、Flt3配体、人白介素-6、粒细胞-巨噬细胞集落刺激因子、粒细胞集落刺激因子、血小板生成素为PeproTec产品，SPF级裸鼠购自北京医科大学动物中心。
方法：构建逆转录病毒载体MGI-F2JAK2，内含有JAK2基因的功能催化区和两个与小分子靶向基因合成药物(AP20187)结合的位点蛋白(2xF36v，F2)组成。AP20187可与F36v特异结合引起JAK2二聚化而激活细胞内信号传导。该载体同时含有绿色荧光蛋白报告基因，用作检测细胞增殖的标记。应用MiniMACS免疫磁珠分选系统纯化分离脐血CD34+ 细胞，用含JAK2的逆转录病毒上清转染脐血CD34+细胞。转导后的CD34+ 细胞在集落刺激因子、Flt3配体、血小板生成素、白介素-6细胞因子的联合培养条件下，以不加或加入AP20187分别作为对照组和实验组。
主要观察指标：①应用流式细胞仪测定两组CD34+细胞中所含绿色荧光蛋白细胞的百分率，确定基因转移率。②扩增后的脐血祖细胞集落培养结果。③取培养10周的脐血CD34+细胞于裸鼠的胁部皮下注射，30 d后观察成瘤情况。
结果：①实验组与对照组均可获得CD34+ 细胞大量扩增。随着培养时间的延长，实验组扩增的CD34+细胞GFP阳性率由基线水平逐渐上升于第11周时达到95%以上，而对照组绿色荧光蛋白报告基因阳性率逐渐下降到基线水平以下并逐渐消失。②实验组转基因CD34+ 细胞于12周后仍可产生造血祖细胞集落红系祖细胞、粒单系祖细胞、脐血多向造血祖细胞，所形成的造血祖细胞集落以粒单系祖细胞为主。③裸鼠实验无致瘤特性。
结论：转染JAK2 基因的人脐血CD34+ 细胞协同其他细胞因子可以体外长期扩增脐血干祖细胞，对今后开展干细胞治疗某些遗传性血液病有潜在的应用价值。
关键词：酪氨酸激酶JAK2；基因治疗；脐血CD34+细胞；扩增

中图分类号: R394.2 文献标识码: A 文章编号: 1673-8225(2008)08-01563-05
赵声明，彭明婷，顾惜春，常乃柏.靶基因调控的脐血干/祖细胞体外长期扩增与调控[J].中国组织工程研究与临床康复，2008，12 (8):1563-1567
[www.zglckf.com/zglckf/ejournal/upfiles/08-8/8k-1563(ps).pdf]
(Edited by Kazuhito Satomura/Ji H/Wang L)